Intercellular recognition complexes formed by T cell receptors (TCR) on cytotoxic T lymphocytes or T helper cells and MHC/peptide complexes on antigen presenting cells (APC) are a common recognition component in a diverse set of cell-cell encounters that activate the TCR both during development of the repertoire of T cells within an individual organism (positive selection; negative selection; peripheral survival) and during the control (T helper) and effector stages (T killer) of an adaptive immune response.
In the adaptive immune response, antigens are recognized by hypervariable molecules, such as antibodies or TCRs, which are expressed with sufficiently diverse structures to be able to recognize any antigen. Antibodies can bind to any part of the surface of an antigen. TCRs, however, are restricted to sensing the presence of antigens by binding to short peptides from the antigens that are presented on the surface of APCs bound to class I or class II molecules of the major histocompatibility complex (MHC). TCR recognition of peptide/MHC complexes triggers activation (clonal expansion) of the T cell.
TCRs are heterodimers composed of two chains which can be αβ (alpha-beta) or γδ (gamma-delta). The structure of TCRs is very similar to that of immunoglobulins (Ig). Each chain has two extracellular domains, which are immunoglobulin folds. The amino-terminal domain is highly variable and called the variable (V) domain. The domain closest to the membrane is the constant (C) domain. These two domains are analogous to those of immunoglobulins, and resemble Fab fragments. The V domain of each chain has three complementarity determining regions (CDR). Proximal to the membrane, each TCR chain has a short connecting sequence with a cysteine residue that forms a disulfide bond between both chains.
Genes encoding αβ and γδ heterodimers are only expressed in the T-cell lineage. The four TCR loci (α, β, γ, and δ) have a germ-line organization very similar to that of Ig. α and γ chains are produced by rearrangements of V and J segments whereas β and δ chains are produced by rearrangements of V, D, and J segments. The gene segments for TCR chains are located on different chromosomes, except the δ-chain gene segments that are between the V and J gene segments of the α chain. The location of δ-chain gene segments has a significance: a productive rearrangement of α-chain gene segments deletes C genes of the δ-chain, so that in a given cell the αβ heterodimer cannot be co-expressed with the γδ receptor.
In mice, there are about 100 Vα and 50 Jα genes segments and only one Cα segment. The δ-chain gene family has about 10 V, 2 D, and 2 J gene segments. The β-chain gene family has 20-30 V segments and two identical repeats containing one Dβ, six Jβ and one Cβ. Finally, the γ-chain gene family contains 7 V and three different J-C repeats. In humans the organization is similar to that of mice, but the number of segments varies.
The rearrangements of gene segments in α and β chains is similar to that of Igs. The α chain, like the light chain of Ig is encoded by V, J, and C gene segments. The β chain, like the heavy chain of Ig, is encoded by V, D, and J gene segments. Rearrangements of a chain gene segments result in VJ joining and rearrangements of β chain result in VDJ joining. After transcription of rearranged genes, RNA processing, and translation, the α and β chains are expressed linked by a disulfide bond in the membrane of T cells.
TCR gene segments are flanked by recognition signal sequences (RSS) containing a heptamer and a nonamer with an intervening sequence of either 12 nucleotides (one turn) or 23 nucleotides (two turn). As in Igs, enzymes encoded by recombination-activating genes (RAG-1 and RAG-2) are responsible for the recombination processes. RAG1/2 recognize the RSS and join V-J and V-D-J segments in the same manner as in Ig rearrangements. Briefly, these enzymes cut one DNA strand between the gene segment and the RSS and catalyze the formation of a hairpin in the coding sequence. The signal sequence is subsequently excised.
The combinatorial joining of V and J segments in a chain and V, D and J segments in β chain produces a large number of possible molecules, thereby creating diversity of TCRs. Diversity is also achieved in TCRs by alternative joining of gene segments. In contrast to Ig, β and δ gene segments can be joined in alternative ways. RSS flanking gene segments in β and δ gene segments can generate VJ and VDJ in the β chain, and VJ, VDJ, and VDDJ on the δ chain. As in the case of Ig, diversity is also produced by variability in the joining of gene segments.
Hypervariable loops of the TCR known as complementarity determining regions (CDRs) recognize the composite surface made from a MHC molecule and a bound peptide. The CDR2 loops of α and β contact only the MHC molecule on the surface of APC, while the CDR1 and CDR3 loops contact both the peptide and MHC molecule. Compared with Ig, TCRs have more limited diversity in the CDR1 and CDR2. However, diversity of CDR3 in TCRs is higher than that of Ig, because TCRs can join more than one D segment leading to augmented junctional diversity.
The pathogenesis of a number of autoimmune diseases is believed to be caused by autoimmune T cell responses to self-antigens present in the organism. Not all autoreactive T cells are deleted in the thymus, in contradiction with the clonal selection paradigm. Those T cells with TCRs for a broad spectrum of self-antigens represent part of the normal T-cell repertoire and naturally circulate in the periphery. It is unclear why autoreactive T cells are allowed, during their evolution, to undergo differentiation in the thymus and are accommodated in the periphery. While their physiological role is not understood, these autoreactive T cells, when activated, present a potential risk in the induction of autoimmune pathologies. Autoreactive T cells can also be isolated from normal individuals without the consequences of autoimmune diseases. It has been established that antigen recognition of autoreactivity by itself is not sufficient to mediate the autodestructive process. One of the prerequisites for autoreactive T cells to be pathogenic is that they must be activated.
Autoreactive T cells are indicated in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis (RA). The pathogenesis of autoreactive T cells in MS is generally held to arise from T cell responses to myelin antigens, in particular myelin basic protein (MBP). MBP-reactive T cells are found to undergo in vivo activation, and occur at a higher precursor frequency in blood and cerebrospinal fluid in patients with MS as opposed to control individuals. These MBP-reactive T cells produce Th1 cytokines, e.g. IL-2, TNF, and γ-interferon, which facilitate migration of inflammatory cells into the central nervous system and exacerbate myelin-destructive inflammatory responses in MS.
MBP-reactive T cells have also been shown to be involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) in animals, which resembles multiple sclerosis. EAE can be induced actively in susceptible animals by injecting MBP emulsified in an adjuvant or passively by injecting MBP-reactive T-cell lines and clones derived from MBP-sensitized animals. When activated in vitro, very small numbers of MBP-reactive T cells are required to induce EAE, while 100-fold more resting T cells with the same reactivity are incapable of mediating the diseases.
It has been demonstrated that vaccination with inactivated MBP-reactive T cells depletes MBP-reactive T cells in EAE, a procedure called T-cell vaccination, which has been used to prevent and to treat EAE. Ben-Nun et al., Nature 292: 60-61 (1981). Although the mechanism underlying T cell vaccination is not completely understood, it is thought to involve the idiotypic regulatory networks through interactions with the TCR and the so-called anti-ergotypic T cell responses that react presumably with the T cell activation markers. The idiotypic and anti-ergotypic regulatory networks are believed to be essential for the protective immunity induced by T cell vaccination, because the protective immunity conferred by T cell vaccination is specific for the disease that autoimmune T cells used for vaccination are able to induce. In addition, anti-idiotypic T cells isolated form immunized rodents specifically recognize the immunizing T cell clones/lines but not T cells expressing distant TCR structural features. It is believed that TCR determinants recognized by anti-idiotypic T cells most likely reside within CDR3 or CDR2, as predicted by characteristic sequence diversity within these regions.
In EAE, encephalitogenic MBP-reactive T cells are restricted to very limited epitopes on MBP. These restrictions in the diversity of the pathogenic T-cell responses permit specific immune intervention. Various therapeutic strategies have been designed accordingly to target the Vβ region of the TCR in preventing the development of EAE in sensitized animals. For example, monoclonal antibodies have been targeted to the Vβ gene product and peptide vaccines have been based on the CDR2 region of the responsible Vβ gene.
Some of the studies on EAE have been extended to human autoimmune diseases. For instance, a peptide corresponding to TCR Vβ 5.2 has been used in clinical trials to treat patients with MS and a Vβ 14 peptide has been used to vaccinate patients with RA. U.S. Pat. No. 5,614,192 (Vandenbark) discloses treatment of autoimmune diseases by the use of immunogenic TCR peptides of 15 to 30 amino acids comprising at least part of CDR2. U.S. Pat. No. 6,303,314 (Zhang) discloses the treatment of autoimmune diseases by using certain immunogenic TCR peptides in combination with immunogenic T cell activation marker peptides.
One area in which vaccination with TCR peptides can be improved is by determining which, if any, common motifs are found in the autoreactive TCRs of a patient with an autoimmune disease such as MS. Such common motifs can be used either as a basis for a peptide vaccine to activate an anti-idiotypic immune response in MS patients for the purpose of depleting T cells which have TCRs comprising said motifs, or as a target for the preparation of antibodies that can functionally block or directly deplete T cells which have TCRs comprising said motifs.
Therefore, it is desirable to determine the amino acid sequences of common motifs specifically found in the TCRs of autoreactive T cells from patients with autoimmune diseases. It is also desirable to be able to readily detect such motifs in a patient sample by a convenient method, such as PCR. In addition, it is desirable to use peptides identical to or derived from the detected motifs to treat patients with the autoimmune disease. It is also desirable to use antibodies which specifically bind to said motifs to treat a patient with an autoimmune disease.
U.S. Pat. No. 6,303,314 (Zhang) discloses such a common motif found in the TCRs of a subset of Vβ13.1 T cells, the “LGRAGLTY motif”, which has the amino acid sequence Leu Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO:10), as well as a method for its ready detection by PCR. This motif is found in some TCRs of some autoreactive T cells that recognize amino acids 83-99 of MBP (hereinafter “MBP83-99”). Peptides based on the LGRAGLTY motif can be used to vaccinate some patients in order to treat or prevent autoimmune diseases with which Vβ13.1-LGRAGLTY is associated (e.g., MS).
As the LGRAGLTY motif is present in only some TCRs that recognize MBP83-99, there remains a need to identify other TCR sequences and more particularly CDR sequences commonly expressed by MBP-reactive T cells. In addition, there remains a need to be able to detect other TCR sequences, including CDR sequences, which are commonly expressed by MBP-reactive T cells. Finally, there remains a need to develop treatments for autoimmune diseases associated with other TCR sequences, and more particularly CDR sequences.